Field of Invention
The subject matter disclosed herein provides immunogenic compositions containing recombinant proteins capable of presenting all, or antigenic portions of, the Eimeria tenella Elongation Factor 1 alpha, or EF-1α, protein to a recipient, such as poultry. The immunogenic compositions are capable of inducing active immunity to, and control of, coccidiosis. Also provided are methodologies of using the immunogenic compositions for administration to poultry and other animals in the control of coccidiosis. In some instances, the EF-1α protein utilized in the immunogenic compositions presented herein is molecularly manipulated or combined with adjuvants to increase effectiveness.
Background
Avian coccidiosis is caused by multiple species of the genus Eimeria and imposes a great economic impact on poultry industry worldwide (Yin et al., Int. J. Parasitol. (2011) 41:813-6; Shirley et al., Avian Pathol. (2012) 41:111-21; Wu et al., Avian Dis. (2014)58:367-72; Lillehoj et al., in “Intestinal Health: Key to Maximize Growth Performance in Livestock”, ed. T. Niewold, (2015) pp. 71-116). Although traditionally, coccidiosis control was successful using prophylactic chemotherapy, increasing concerns with drug resistance, drug residue and the restricted governmental regulation on the use of drugs in agricultural animals hinder its application (Jeffers, J. K., in “Coccidia and Intestinal Coccidiomorphs”, ed. P. Yvore (1989) pp 295-308; Lillehoj et al., Poultry Sci. (2007) 86:1491-1500; Lin et al., Gene (2011) 480:28-33). Immunization is an effective and cost-effective method of preventing infection and a live coccidiosis vaccine has been used for more than 50 years. However, the live vaccine is not widely used, most likely due to the risk of unintended infection, and inconsistent immunity development causes by many different clinical factors such as climate and management (Wu et al., supra). Additionally, live coccidiosis vaccines consist of multiple different species of Eimeria, even different strains in some species of Eimeria spp. to account for the varied immunogenicity (Smith et al., Infect. Immun. (2002) 70:2472-9; Allen et al., Parasitol. Res. (2005) 97:179-85).
In recent years, induction of protective immunity using peptide vaccines has gained much interest with increasing technological advances in genetic engineering and protein expression (Shirley et al., supra; Lillehoj et al., supra). Immunogenic proteins from various stages of Eimeria have been tried with various levels of success and when combined with mucosal delivery adjuvants, or components that enhance cell-mediated immunity, significant protective immune responses that decrease negative consequences of coccidiosis were reported (Lillehoj et al., supra). However, there remains an inability to elicit optimal levels of protective response against multiple coccidia species due to their weak immunogenicity and poor/undetermined cross-protection against different species. Thus, many challenges still remain before peptide antigens can be applied in commercial poultry production (Jang et al., Vaccine (2010) 28:2980-5; Shirley et al., supra; Liu et al., Parasit. Vectors (2014) 7:27; Xu et al., Korean J. Parasitol. (2013) 51:147-54).
The phylum Apicomplexa, which includes species of the genus Eimeria, comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexans (referred as zoites) including Cryptosporidium spp., possess a unique complex of organelles located at the anterior end of the organism (the apical complex). The apical complex comprises rhoptries, micronemes, dense granules, and an apical assembly of cytoskeleton-associated structures such as the conoid, polar/apical rings, and microtubular protrusions. The apical complex of zoites of Cryptosporidium spp. (Lumb et al., Parasitol. Res. (1988) 74:531-6; Hamer et al., Infect. Immun. (1994) 62:2208-13; Riggs et al., Infect. Immun. (1999) 67:1317-22; Schaefer et al., Infect. Immun. (2000) 68:2608-16) and other closely related Apicomplexans (Tomley et al., Mol. Biochem. Parasitol. (1996) 79:195-206; Brown and Palmer, Parasitol. Today (1999) 15:275-81; Carruthers et al., Cell. Microbiol. (1999) 1:225-35; Lovett et al., Mol. Biochem. Parasitol. (2000) 107:33-43; Hu et al., J. Cell Biol. (2002) 156:1039-50) are involved in parasite attachment, invasion, and intracellular development. Thus, these organelles and their molecular constituents are thought to provide rational targets for immunological therapy or drug treatment to control infections by these parasites.
In Eimeria, very limited information on conserved proteins that elicit protective immune response against multiple species of Eimeria has been reported (Lillehoj et al., supra). Elongation Factor-1α (“EF-1α”) is highly conserved and ubiquitously expressed in all eukaryotic cells (Riis et al., Trends Biochem. Sci. (1990) 15:420-4). Previous studies have revealed that EF-1α regulates protein synthesis and plays an important role in the progress of invasion of host-cells by Apicomplexan parasites (Abrahamsen et al., Mol. Biochem. Parasitol. (1993) 57:1-14; Amiruddin et al., BMC Genomics (2012) 13:21; Matsubayashi et al., J. Biol. Chem. (2013) 288:34111-20).
Although immunogenic Eimeria proteins have yet to be proven in commercial applications against coccidiosis, recent studies on expressed recombinant proteins have shown various levels of protective immune response against Eimeria challenge with some examined parameters, which promoted the development of recombinant vaccines against coccidiosis (Jang et al., supra; Ding et al., Parasitol. Res. (2012) 110:2297-306; Liu et al., Parasitol. Res. (2013) 112:251-7; Zhao et al., Parasitol. Res. (2014) 113:3007-14). Usually, avian coccidiosis is caused by multiple different species of the genus Eimeria, which are antigenically distinct and have complex life cycles, thus the identification and application of more and highly conserved protective epitopes will be helpful for the control of different Eimeria species.
As described herein, we carried out experiments to clone the EF-1α gene from E. tenella, express EF-1α recombinant protein, and evaluate its immunogenicity and protective efficacy against E. tenella challenge infection in commercial broiler chickens. To do this, we constructed a prokaryotic plasmid pET-EF1α, expressed and purified the rET-EF1α and evaluated its efficacy against E. tenella or E. maxima. The results show that rEF-1α from E. tenella can elicit cross protective immunity against other species of Eimeria. 